Dimeric contrast agents

ABSTRACT

The present invention relates to new class of dimeric macrocycles capable of chelating paramagnetic metal ions, their chelated complexes with metal ions and the use thereof as contrast agents, particularly suitable for Magnetic Resonance Imaging (MRI) analysis.

FIELD OF THE INVENTION

The present invention relates to the field of diagnostic imaging and tonovel contrast agents possessing improved relaxivity. More inparticular, it relates to dimeric macrocycles capable of chelatingparamagnetic metal ions, their chelated complexes with metal ions andthe use thereof as contrast agents in Magnetic Resonance Imaging (MRI).

STATE OF THE ART

Magnetic Resonance Imaging (MRI) is a renowned diagnostic imagingtechnique increasingly used in clinical diagnostics for growing numberof indications.

The undisputed success of this technique is determined by the advantagesit offers, including a superb temporal and spatial resolution, theoutstanding capacity of differentiating soft tissues and its safety, dueto its non-invasiveness and the absence of any ionizing radiation, incontrast to, for instance, X-ray, PET and SPECT.

In MRI imaging the contrast is basically due to differences existing inthe longitudinal T1 and the transverse T2 relaxation times of the waterprotons in the different body organs and tissues, which allows thein-vivo acquisition of high-resolution, three-dimensional images of thedistribution of water.

The intensity of the signal recorded in MRI imaging stems, essentially,from the local value of the longitudinal relaxation rate 1/T1, and thetransverse rate, 1/T2 of water protons, and increases with increasing ofthe 1/T1 value (of the longitudinal relaxation rate of water protons)while decreases with the increase of 1/T2. In other words, the shorteris T1, the higher is the intensity of the recorded signal in MRI, thebetter is the diagnostic image.

The strong expansion of medical MRI has further benefited from thedevelopment of a class of compounds, the MRI contrast agents, that actby causing a dramatic variation of nearby water proton relaxation ratesin the tissues/organs/fluids wherein they distributes, thus addingrelevant physiological information to the impressive anatomicalresolution commonly obtained in the uncontrasted MRI images.

Contrast agents used in the MRI imaging technique typically include aparamagnetic metal ion which is complexed with a cyclic or acyclicchelating ligand, more typically a polyaminopolycarboxylic chelator. Themost important class of MRI contrast agents is represented by theGd(III) chelates which are currently used in about ⅓ of the clinicaltests. Indeed, Gd(III) is highly paramagnetic with seven unpairedelectrons and a long electronic relaxation time, making it an excellentcandidate as a relaxation agent. On the other hand, the free metal ion[Gd(H₂O)₈]³⁺ is extremely toxic for living organism even at low doses(10-20 micromol/Kg). Thus, in order to be considered as a potentiallyvaluable MRI contrast agent, a Gd(III) complex shall display a highthermodynamic (and possibly kinetic) stability in order to prevent therelease of toxic metal ion.

Preferred MRI contrast agent should furthermore display optimalrelaxivity. Relaxivity (r_(1p), r_(2p)), expressed in mM⁻¹ s⁻¹ andusually measured at 298K and 20 MHz (approx. 0.5 T), is the intrinsicproperty of a paramagnetic complex which characterizes its capability toincrease the nuclear magnetic relaxation rate, longitudinal (1/T₁) andtransverse (1/T2) respectively, of vicinal water protons and, thus, itsefficacy as MRI contrast enhancing agent. In general terms, the higherthe relaxivity of an MRI contrast agent, the greater its contrastenhancing capability and the stronger the contrast provided in recordedMRI images.

A number of complexes of paramagnetic metal ions are known in the art(see for instance: Caravan P. et al. Chem. Rev. 1999, 99, 2293-2352 andU.S. Pat. Nos. 4,647,447, 4,885,363; 4,916,246; 5,132,409; 6,149,890;and 5,980,864).

Dimeric complexes are disclosed for instance in U.S. Pat. No. 5,277,895,DE10117242, and DE19849465.

Examples of commercially available MRI contrast agents include thecomplex compound of the Gd³⁺ ion with the DTPA ligand, marketed asMAGNEVIST®; the Gd³⁺ complex of the DTPA-BMA ligand, marketed asOMNISCAN®; the Gd³⁺ complex of BOPTA, known as gadobenate Dimeglumineand marketed as MultiHance™; the Gd³⁺ complex of the DOTA ligand,marketed as DOTAREM®; the Gd³⁺ complex of the hydroxylated tetraazamacrocyclic ligand known as HPDO3A, long time marketed as ProHance® andthat of the corresponding butyl-triol derivative, known as Gadobutroland marketed ad Gadavist®. All the above contrast agents comprise asingle chelating unit, and are Non-Specific Agents (NSA), designed for ageneral use.

While known compounds generally provide a quality of the imaging capableof meeting and satisfying the present needs of radiologists resulting inaccurate and detailed diagnostic information, there is neverthelessstill the need for new compounds with improved contrast imagingfeatures, such as increased relaxivity.

In particular, compounds with improved relaxivity could reduce therequired dose of the paramagnetic contrast agent and possibly shortenthe acquisition time of the imaging process.

SUMMARY OF THE INVENTION

The present invention generally relates to novel macrocyclic chelatingligands useful for the preparation of paramagnetic complexes havingparticularly favorable characteristics, among others in terms ofimproved relaxivity.

In general terms, an aspect of the present invention relates to noveldimeric ligands comprising two tetraaza macrocycles with a hydroxylatedresidue on a nitrogen atom of the chelating cage linked to one anotherthrough amine group(s).

The invention further relates to respective chelated complexes of saidchelating ligands with a paramagnetic metal ion and, especially, withGd³⁺, or of a physiologically acceptable salt thereof.

A further aspect of the invention relates to the use of such chelatedcomplexes as contrast agents, in particular for the diagnostic imagingof a human or animal body organ or tissue by use of the MRI technique.

In a further aspect the invention relates to a manufacturing process forthe preparation of the provided ligands, their complex compounds with aparamagnetic metal ion, and the pharmaceutical acceptable salt thereofand their use in the preparation of a diagnostic agent.

According to another aspect, the invention relates to a pharmaceuticallyacceptable composition comprising at least one paramagnetic complexcompound of the invention, or a pharmaceutical salt thereof, inadmixture with one or more physiologically acceptable carriers orexcipients. Said compositions are useful in particular as MRI contrastmedia, to provide diagnostically useful images of human or animal bodyorgans or tissues.

Therefore, in another aspect, the present invention refers to a methodfor the diagnostic imaging of a body organ, tissue or region by use ofMRI technique that comprises the use of an effective dose of a compoundof the invention.

DETAILED DESCRIPTION OF THE INVENTION

An object of the present invention are chelating ligands of formula (I)

where:

-   -   R is —CH(R₁)—COOH, where:        -   R₁ is H or a C₁-C₃ alkyl chain that is optionally            substituted by a C₁-C₃ alkoxy or C₁-C₃ hydroxyalkoxy group;    -   n is 1 or 2;    -   R₂ is selected from the group consisting of: an aryl ring; a        cycloalkyl ring; a C₁-C₅ alkyl substituted by one ore more C₁-C₈        hydroxyalkoxy groups, or by a cycloalkyl ring; a group of        formula —(CH₂)_(s)CH(R₃)-G; and a C₅-C₁₂ hydroxyalkyl comprising        at least 2 hydroxyl groups;    -   in which        -   s is 0, 1 or 2;        -   G is a group selected from —PO(OR₄)₂, —PO(R₅)(OR₄) and            —COOH;        -   R₃ is H, or an arylalkylene or cycloalkyl-alkylene having            from 1 up to 3 carbon atoms in the alkylene chain;        -   R₄ independently of one another is H or C₁-C₅alkyl;        -   R₅ is an aryl or cycloalkyl ring, or C₁-C₅ alkyl which is            optionally substituted by an aryl or cycloalkyl ring; and    -   L is a C₁-C₆ alkylene, optionally interrupted by one or more        —N(R′₂)— groups, and optionally substituted by one or more        substituent groups selected from hydroxyl, C₁-C₃ alkoxy and        C₁-C₃ hydroxyalkoxy, where    -   R′₂ is, independently, as defined for R₂.

Preferably in the above compounds of formula (I) R₁ is H.

In the present description, and unless otherwise provided, theexpression “alkyl” comprises within its meaning any linear or branchedhydrocarbon chain, preferably comprising up to 12 carbon atoms. Inparticular “C₁-C₁₂ alkyl” comprises within its meaning a linear orbranched chain comprising from 1 to 12 carbon atoms such as: methyl,ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, pentyl,iso-pentyl, tert-pentyl, hexyl, iso-hexyl, heptyl, iso-heptyl, octyl,and the like. Similarly, the term “C₁-C₃ alkyl” comprises within itsmeaning a linear or branched chain comprising from 1 to 3 carbon atomssuch as, for instance, methyl, ethyl, propyl and iso-propyl; the term“C₁-C₆ alkyl” comprises within its meaning a linear or branched chaincomprising from 1 to 6 carbon atoms such as: methyl, ethyl, propyl,iso-propyl, butyl, iso-butyl, tert-butyl, pentyl, hexyl and the like;and the term “C₅-C₇ alkyl” comprises within its meaning any linear orbranched chain comprising from 5 to 7 carbon atoms such as pentyl,iso-pentyl, tert-pentyl, hexyl, iso-hexyl, tert-hexyl, heptyl,iso-heptyl and tert-heptyl.

By analogy, the expression “alkylene” comprises within its meaning abivalent linear or branched chain derived by any of the abovehydrocarbon chains by removal of two hydrogen atoms from differentcarbon atoms, e.g. including C₁-C₆ alkylene such as for instance amethylene, ethylene, (iso)propylene and so on.

The term “hydroxyalkyl” comprises within its meaning any of thecorresponding alkyl chain wherein one or more hydrogen atoms arereplaced by hydroxyl groups. Suitable examples include C₁-C₃hydroxyalkyl such as hydroxymethyl (—CH₂OH), hydroxyethyl (—CH₂CH₂OH),hydroxypropyl (—CH₂CH₂CH₂OH), dihydroxypropyl, (—CH₂CH₂OHCH₂OH and—CH(CH₂OH)₂) and the like, and polyhydroxyalkyls or “polyols”, as usedherein interchangeably, in which at least two and, preferably, three ormore hydrogen atoms of the hydrocarbon chain are replaced by hydroxylgroups.

For instance, and unless otherwise provided, the expression “C₅-C₁₂polyol” (or “C₅-C₁₂ polyhydroxyalkyl”) comprises within its meaning anyof the corresponding C₅-C₁₂ alkyl moiety in which 2 or more, e.g. from 2to 11 hydrogen atoms have been replaced by hydroxyl groups. Among them,C₅-C₁₀ polyols are preferred, and C₅-C₇ polyols are particularlypreferred. Examples of C₅-C₇ polyols include pentyl-polyols (orpolyhydroxypentyls) such as pentyl-diols, pentyl-triols, pentyl-tetraolsand pentyl-pentaol, respectively comprising from 2, 3, 4 and 5 hydroxylgroups on a C₅ alkyl chain; hexyl-polyols (or polyhydroxyhexyls)analogously comprising from 2 to 6 hydroxyl groups on a C₆ alkyl chain;and heptyl-polyols (or polyhydroxyheptyls) comprising from 2 to 7hydroxyl groups on a C₇ alkyl chain.

The term “alkoxy” comprises within its meaning an alkyl chain as abovedefined further comprising one or more oxygen atoms; examples include,for instance, alkyl-oxy (or —Oalkyl) groups such as methoxy, ethoxy,n-propoxy, isopropoxy and the like, and alkyl-(poly)oxy in which thealkyl chain is interrupted by one or more, e.g. up to three, oxygenatoms.

The term “hydroxyalkoxy” comprises within its meaning any of the abovealkyloxy residues further comprising one or more hydroxyl (—OH) in thealkyl chain such as, for example, —OCH₂OH, —OCH₂CH₂OH, —OCH₂CH₂CH₂OH,—OCH₂OCH₂OH, —OCH₂CH₂OCH₂CH₂OH, —OCH₂CH(OH)CH₂—OCH₂CH₂OH, and the like.

The term “hydroxyalkoxyalkylene” (or “hydroxyalkoxy-alkylene”) compriseswithin its meaning any of the above hydroxyalkoxy where the linkinggroup of the residue is an alkylene chain —(CH₂)_(r)—, including C₂-C₁₀hydroxyalkoxy-alkylenes e.g. of formula—(CH₂)_(r)—[(O—(CH₂)_(r)]_(r)(CH₂)_(s)OH, where each r is independently1 or 2, and s is 0, 1 or 2.

The expression “carboxyl” comprises within its meaning a residue offormula —COOH, or comprising said —COOH residue, such as the groups offormula —(CH₂)_(s)—COOH or —[(O(CH₂)_(n)]_(s)—COOH, where s and n are asabove defined.

The term “aryl” or “aryl ring” refers to an aromatic hydrocarbon and,preferably, a phenyl ring. Unless otherwise specifically provided, arylsaccording to the invention can be either unsubstituted or substitutedwith one or more, equal or different, substituent groups, for instanceselected from hydroxyl (OH), halogen, C₁-C₃ alkyl, C₁-C₃ alkoxy, C₁-C₃hydroxyalkyl, carboxy, carbamoyl, nitro, —NH₂, or C₁-C₃ alkyl- ordialkylamino, preferably from hydroxyl, halogen, C₁-C₃ alkyl or alkoxy,or carboxy and, more preferably, from C₁-C₃ alkyl or alkoxy, —CH₂COOH,and —COOH.

The term “cycloalkyl ring” (or “cycloalkyl”) as used herein compriseswithin its meaning a saturated (i.e. cycloaliphatic), either carbocyclicor heterocyclic ring.

Suitable examples include a C₅-C₇ carbocyclic ring e.g. a cyclohexylring. Unless otherwise specifically provided, carbocyclic ringsaccording to the invention can be either unsubstituted or substitutedwith one or more, equal or different, substituent groups for instanceselected from hydroxyl halogen, C₁-C₃ alkyl, C₁-C₃ alkoxy, C₁-C₃hydroxyalkyl, carboxyl, carbamoyl, nitro, —NH₂, or C₁-C₃ alkyl- ordialkylamino, preferably from hydroxyl, halogen, C₁-C₃ alkyl or alkoxyor carboxy and, more preferably, from C₁-C₃ alkyl or alkoxy, —CH₂COOH,and —COOH.

“Cycloalkyl ring” according to the invention further include a saturatedheterocyclic ring (or heterocycle) e.g., preferably, a 5-6 memberedsaturated ring comprising a nitrogen atom in the cyclic chain and,optionally, another, equal or different, heteroatom selected from N, Oand S. Suitable examples include heterocycles such as pyrrolidine,piperazine, morpholine and piperidine, wherein this last is particularlypreferred. Nitrogen-containing heterocycles according to the inventionpreferably comprise one or more substituents groups linked to the carbonatom(s) of the cycle, e.g. selected from hydroxyl, C₁-C₃ hydroxyalkyl,C₁-C₃ alkoxy, C₁-C₃ hydroxyalkoxy, C₁-C₃ hydroxyalkoxy-alkyl, and acarboxyl such as —(CH₂)_(s)—COOH or —[(O(CH₂)_(n)]_(s)—COOH, as abovedefined.

From all the above, having defined the meaning for alkyl, alkylene, aryland cycloalkyl, any composite-name such as alkyl-aryl, aryl-alkylene,cycloalkyl-alkylene and the like should be clear to a skilled person.

For instance the term alkylaryl (or alkyl-aryl) comprises within itsmeaning an aryl group further substituted by an alkyl, (e.g.p-ethyl-phenyl; pC₂H₅—C₆H₅—) while the term arylalkylene (oraryl-alkylene) or cycloalkyl-alkylene comprises within its meaning analkyl further substituted by an aryl (e.g. phenyl-ethylene=C₆H₅—C₂H₄—)or by a cycloalkyl (e.g. cyclohexyl-ethylene=C₆H₁₁—C₂H₄—); and the like.

In the present description the term “protecting group” designates aprotective group adapted for preserving the function of the group towhich it is bound. Specifically, protective groups are used to preserveamino, hydroxyl or carboxyl functions. Appropriate carboxyl protectivegroups may thus include, for example, benzyl, alkyl e.g. tert-butyl orbenzyl esters, or other substituents commonly used for the protection ofsuch functions, which are all well known to those skilled in the art[see, for a general reference, T. W. Green and P. G. M. Wuts; ProtectiveGroups in Organic Synthesis, Wiley, N.Y. 1999, third edition].

Moreover, the terms “moiety” or “moieties”, “residue” or “residues” areherewith intended to define the residual portion of a given moleculeonce properly attached or conjugated, either directly or through anysuitable linker, to the rest of the molecule.

The compounds of the above formula (I) may have one or more asymmetriccarbon atom, otherwise referred to as a chiral carbon atom, and may thusgive rise to diastereomers and optical isomers. Unless otherwiseprovided, the present invention further includes all such possiblediastereomers as well as their racemic mixtures, their substantiallypure resolved enantiomers, all possible geometric isomers, andpharmaceutical acceptable salts thereof.

The present invention further relates to compounds of the above formula(I) in which each of the acidic groups, either including the carboxylicgroups R linked to the nitrogen atoms of the macrocycles or any otheroptional acidic group, e.g. on R₂, may be in the form of apharmaceutically acceptable salt, or of a derivative in which the acidicgroup is suitably protected with an appropriate protecting group (Pg) asabove defined, e.g., preferably, of a C₁-C₅ alkyl ester and, morepreferably, of a tert-butyl ester, finding for instance application assuch, or as suitable precursor or intermediate compound in thepreparation of a desired compound of formula (I) or of a suitableparamagnetic complex or salt thereof.

In one embodiment, the invention relates to dimeric compounds of formula(I) in which L is a C₁-C₆ alkylene chain.

Suitable examples include dimers of formula (II)

in which:

n is 1 or 2;

m is 1, 2, 3, 4, 5 or 6; and

R₂ is as defined for compounds of formula (I).

In one embodiment, in the above compounds of formula (II) R₂ is an arylor a cycloalkyl ring, e.g., preferably, a phenyl or a cyclohexyl ring.

In another embodiment the invention relates to compounds of formula (II)in which R₂ is a C₅-C₁₂ hydroxyalkyl comprising at least two hydroxylgroups.

Suitable examples include compounds in which in the formula (II) R₂ is aC₅-C₁₂ polyhydroxyalkyl (or C₅-C₁₂ polyol) having from 2 to 11 and,preferably, from 3 to 10 hydroxyl groups on the C₅-C₁₂ alkyl chain.

Preferably, R₂ is the residue of a C₅-C₇ polyol e.g. selected frompentyl-polyols (or polyhydroxypentyls) comprising at least 2, andpreferably from 2 to 4 hydroxyl groups on the C₅ alkyl chain;hexyl-polyols comprising at least 2, and preferably from 2 to 5 hydroxylgroups on the C₆ alkyl chain; and heptyl-polyols comprising at least 2and, and preferably from 3 to 6 hydroxyl groups on the C₇ alkyl chain.

In particular, in one preferred embodiment the invention relates tocompounds of formula (II A)

in which P is a C₅-C₇ polyol selected from a pentyl-tetraol of formula

and a hexyl-pentaol of formula

and n and m are as defined for compounds of formula (II).

Preferably, in the compounds of formula (II A) n and m, independently toone another, are 1 or 2. More preferably are both 1.

In a particularly preferred embodiment, the invention relates to adimeric compound according to the above formula (II A), having theformula

In a further embodiment, the invention relates to compounds according tothe formula (II) in which R₂ is a group of formula —(CH₂)_(s)CH(R₃)-Gwhere s, R₃ and G are as above defined for compounds of formula (I).

Preferably, in these compounds R₃ is H or an arylalkylene orcycloalkyl-alkylene selected from benzyl, phenyl-ethyl,cyclohexyl-methyl and cyclohexyl-ethyl; and G is a group of formula—PO(OR₄)₂, —PO(R₅)(OR₄) and —COOH, in which R₄ is H or a tert-butyl, andR₅ is selected from an optionally substituted phenyl or cyclohexyl ringand a C₁-C₅ alkyl chain, e.g., preferably, a methyl, ethyl or propylgroup, which is substituted or not by an aryl or cycloalkyl ring such asa benzyl, phenyl-ethyl, cyclohexyl-methyl or cyclohexyl-ethyl group.

More preferably in the above compounds R₃ is H.

In particular, in one preferred embodiment the invention relates tocompounds of formula (II B)

in which:

-   -   s is 0 or an integer from 1 to 2;    -   G is a group selected from —PO(OR₄)₂, —PO(R₅)(OR₄) and —COOH,        where R₄ is as is H or a tert-butyl and, preferably, is H; R₅ is        an optionally substituted phenyl or cyclohexyl ring, or a C₁-C₃        alkyl substituted or not by an aryl or cycloalkyl ring such as        benzyl, phenyl-ethyl, cyclohexyl-methyl or cyclohexyl-ethyl; and    -   m and n are as said for the compounds of formula (II).

In a particularly preferred embodiment, the invention relates tocompounds formula (II B) in which G is selected from —PO(OH)₂ and —COOH;s is 0 or 1; n and m, independently to one another, are 1 or 2 and,preferably, are both 1.

According to an additional embodiment, the invention relates tocompounds of formula (II) in which R₂ is a C₁-C₅ alkyl which issubstituted by one or two C₁-C₈ hydroxyalkoxy groups, or by a cycloalkylring.

In one preferred embodiment R₂ is a C₁-C₅ alkyl substituted by a C₁-C₈hydroxyalkoxy group.

Suitable examples include dimers of formula (II) in which R₂ is a C₂-C₁₀hydroxyalkoxy-alkylene e.g. selected from the groups of formula—CH₂(OCH₂CH₂)_(s)OCH₂OH, —CH₂(CH₂OCH₂)_(r)CH₂OH and—(CH₂)_(r)—O(CH₂)_(r)OH, where r and s are as said.

Preferred among them are compounds of formula (II C)

in which each n, m and r, independently the one another, is an integerfrom 1 to 2.

Particularly preferred are compounds of formula (II C) in which n and mare both 1.

In another embodiment R₂ is a C₁-C₅ alkyl substituted by two C₁-C₈hydroxyalkoxy groups.

Suitable examples include compounds of formula (II) in which R₂ is abranched C₁-C₅ alkyl, e.g. isopentyl or isobutyl, which is substitutedby two C₁-C₈, and, preferably, C₁-C₅ hydroxyalkoxy groups.

Preferably, R₂ is a isopropylen or, more preferably, a isobutylenbearing two terminal polyhydroxyalkoxy groups selected from—OCH₂(CH₂OH)₂ and —OCH₂(CH₂CH₂OH)₂.

In a still further embodiment the invention relates to compounds offormula (II) in which R₂ is a C₁-C₅ alkyl substituted by a cycloalkylring.

Suitable examples include compounds in which R₂ is a C₁-C₅ alkylsubstituted by a saturated C₅-C₇ carbocyclic ring such as a cyclohexylring, e.g., preferably, a cyclohexyl-alkylene having 1, 2 or 3 carbonatoms in the alkylene chain.

More preferably, R₂ is a C₁-C₅ alkyl substituted by a saturated C₅-C₇heterocycle, e.g. a piperidine or a piperidine derivative having one ormore e.g. from 1 to 8 substituents groups linked to the carbon atom(s)of the heterocycle.

In particular, in a further embodiment the invention relates to dimersof formula (II D)

in which

n and m are, each independently, 1 or 2 and, preferably, are both 1;

p is an integer from 1 to 3;

q is

and integer from 1 to 8, and

S is a substituent group linked to a carbon atom of the piperidine ring,e.g. selected from the group consisting of: hydroxyl, C₁-C₃hydroxyalkyl, C₁-C₃ alkoxy, C₁-C₃ hydroxyalkoxy, C₁-C₃hydroxyalkoxy-alkylene, and carboxyl such as —(CH₂)_(s)—COOH and—OCH₂—COOH where s is as above said.

For instance, in one embodiment in the above compounds of formula (II D)q is 1, and S is a group selected from hydroxyl, C₁-C₃ hydroxyalkyl,C₁-C₃ hydroxyalkoxy and carboxyl such as —(CH₂)_(s)—COOH or —OCH₂—COOHand, more preferably, from hydroxyl, —CH₂OH, and —COOH that is linked tothe C₃ carbon atom of the ring.

Preferably, in the above compounds formula (II D) q is an integer from 2to 8, and the compounds comprise a piperidine ring having from 2 to 8,preferably from 2 to 6 and, more preferably, from 3 to 5 e.g. 3, 4, or 5substituent groups S linked to one or more carbon atom(s) of the ring,that are each independently selected from hydroxyl, C₁-C₃ hydroxyalkyl,C₁-C₃ alkoxy, C₁-C₃ hydroxyalkoxy, C₁-C₃ hydroxyalkoxy-alkylene, andcarboxyl such as —(CH₂)_(s)—COOH or —(OCH₂)_(s)—COOH.

According to an alternative embodiment, the invention relates tocompounds according to the formula (I) in which L is a C₁-C₆ alkylenechain interrupted by one or two —N(R′₂)— groups.

Suitable examples include dimeric compounds of formula (III)

in which:

each n, r and d is, independently, 1 or 2; and

R₂ and R′₂ are as defined for the compounds of formula (I).

In one embodiment, in the above formula (III) d is 1, and the inventionrelates to dimers comprising two macrocyclic residues having ahydroxylated pendant arm bound to a nitrogen atom of the chelating cagelinked to one another by means of a diamine group of formula—N(R₂)—(CH₂)_(r)—N(R′₂)—

In one embodiment, in the above compounds of formula (III) R₂ and R₂′,equal of different, are each independently selected from R₂ meanings.

Preferably, in the compounds of formula (III) R₂′ is the same as R₂.

In particular, in one preferred embodiment the invention relates todimeric compounds of formula (IV)

in which each n and r is, independently, 1 or 2, and R₂ is as said forcompounds of formula (II), including encompassed formulae from (II A) to(II D).

Suitable examples include compounds of formula (IV) in which R₂ isselected from the groups of formula —CH₂(OCH₂CH₂)_(s)OCH₂OH,—CH₂(CH₂OCH₂)_(r)CH₂OH and —(CH₂)_(r)—O(CH₂)_(r)OH, in which r and s areas said. Preferably, R₂ is —CH₂(CH₂OCH₂)_(r)CH₂OH, where r is 1 or 2.

According to a more preferred embodiment, in the above formula (IV) R₂is a group of formula —(CH₂)_(s)CH(R₃)-G where s, R₃ and G are asdefined for compounds of formula (I).

Preferably, in these compounds R₃ is H or an arylalkylene orcycloalkyl-alkylene e.g. selected from benzyl, phenyl-ethyl,cyclohexyl-methyl and cyclohexyl-ethyl; G is a group of formula—PO(OR₄)₂, —PO(R₅)(OR₄) and —COOH in which R₄ is H or a tert-butyl and,preferably, is H, and R₅ is an optionally substituted phenyl orcyclohexyl ring, or a C₁-C₃ alkyl such as methyl, ethyl or propylsubstituted or not by an aryl or cycloalkyl ring.

In particular, in one preferred embodiment the invention relates todimers of formula (IV A)

in which n is an integer from 1 to 2 and, preferably is 1;

r is 1 or 2;

s is 0 or an integer from 1 to 2, and preferably is 0 or 1; and

G a group selected from —PO(OR₄)₂ and —COOH where R₄ is H or atert-butyl and, preferably, is H.

More preferably in the compounds of formula (IV A) n is 1, r is 2, and sis 0.

Particularly preferred according to the invention are dimers of formula(IV A) selected from

Particularly preferred compounds are those compounds of formula (I), orsalts thereof, selected from the group consisting of:

In a further aspect the invention relates to chelated complexes of thecompounds of formula (I), hence encompassing those of formulae from (II)to (V), with two paramagnetic metal ions, or radionuclides, or of asuitable salt thereof.

Preferably, the paramagnetic metal ions are are equal to each other, andare selected in the group consisting of Fe²⁺, Fe³⁺, Cu²⁺, Cr³⁺, Gd³⁺,Eu³⁺, Dy^(3+,) La³⁺, Yb³⁺ or Mn²⁺. More preferably, both the cheatedparamagnetic metal ions are Gd³⁺ ions.

Preferred radionuclides according to the invention providing complexesfor use in radiotherapy or radiodiagnostics include ¹⁰⁵Rh, ^(117m)Sn,^(99m)Tc, ^(94m)Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁴⁴Sc, ⁷²As, ¹¹⁰n, ¹¹¹In, ¹¹³In,⁹⁰Y, ⁹⁷Ru, ⁶⁰Cu, ⁶²Cu, ⁶⁴Cu, ⁵²Fe, ⁵¹Mn, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁵³Sm, ¹⁶⁶Ho,¹⁴⁹Pm, ¹⁷⁷Lu, ^(186/188)Re, ¹⁶⁵Dy, ¹⁶⁶Dy, ¹⁴²Pr, ¹⁵⁹Gd, ²¹¹Bi, ²¹²Bi,²¹³Bi, ²¹⁴Bi, ¹⁴⁹Pm, ⁶⁷Cu, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb, ¹⁶⁷Tm, and ⁵¹Cr.

As formerly reported, both the compounds of formula (I) of the inventionand the paramagnetic chelates thereof can also be in the form of apharmaceutically acceptable salt, particularly as an addition salt witha physiologically compatible base or acid.

The term “pharmaceutically acceptable salt”, as used herein, refers toderivatives of the compounds of the invention wherein the parentcompound is suitably modified by converting any of the free acid orbasic groups, if present, into the corresponding addition salt with anybase or acid conventionally intended as being pharmaceuticallyacceptable.

Preferred cations of inorganic bases which can be suitably used toprepare a salt of the complexes or the ligands of the inventioncomprise, for instance, ions of alkali or alkaline-earth metals such aspotassium, sodium, calcium or magnesium.

Preferred cations of organic bases comprise, for instance, those ofprimary, secondary and tertiary amines such as, for instance,ethanolamine, diethanolamine, morpholine, glucamine, N-methylglucamine,N,N-dimethylglucamine.

Preferred anions of inorganic acids which can be suitably used toprepare salts of the complexes of the invention comprise the ions ofhalo acids, for instance chlorides, bromides or iodides, as well as ofother suitable ions such as sulfate.

Preferred anions of organic acids comprise those routinely used inpharmaceutical techniques for the salification preparation of salts ofbasic substances such as, for instance, acetate, succinate, citrate,fumarate, maleate or oxalate.

Preferred cations and anions of amino acids comprise, for instance,those of taurine, glycine, lysine, arginine, ornithine or of asparticand glutamic acids.

The preparation of the compounds of formula (I), hence encompassing thecompounds of formulae from (II) to (IV), and of the chelate complexesthereof, either as such or in the form of physiologically acceptablesalts, represent a further object of the invention.

Compounds of formula (I), and the chelated complexes thereof, may beprepared through a general synthetic process comprising the followingsteps:

-   -   a) Obtaining a macrocyclic substrate 1 in a suitable protected        form, e.g. in which the carboxylic groups of the substrate are        protected as tert-butyl esters;    -   b) Obtaining a bridging molecule 2, in which any optional        functional group(s) not involved with the coupling reaction with        the substrate 1 is, optionally, suitably protected;    -   c) Coupling the bridging molecule 2 with two units of protected        substrate 1, to give the desired compound of formula (I) in a        suitably protected form or, alternatively, an intermediate        thereof 3;    -   d) Optionally converting the obtained intermediate in the        suitably protected compound of formula (I);    -   e) Removing any protecting group and isolating the chelating        ligand of formula (I); and    -   f) Complexing the obtained ligand with a suitable paramagnetic        metal ion and isolating the chelate complex, or the salt        thereof.

To this extent, and unless otherwise indicated, the term “intermediate”(e.g. with reference to the compound 3 deriving from the reaction of themacrocyclic substrate 1 with an bridging molecule 2) refers to amolecule that requires one (or more) further reactions, e.g.deprotection/alkylation reaction(s) converting any optional protectednitrogen atom(s) of the bridging molecule 2 in the correspondingalkylated derivative(s), to give the desired product, i.e. in thespecific case of the above general scheme, in a suitably protecteddimeric compound of formula (I) according to step d). The single stepsof the above general process, comprehensive of any variant thereof,particularly when referring to the steps of protection/deprotection andactivation of known functional groups, may be carried out according toconventional methods known in the art.

For instance, suitable substrates 1A according to the step a) of theprocess of the invention, of formula

in which all carboxyl groups are suitably protected as tert-butylesters, may be obtained e.g. as disclosed in Org. Synth. 2008, 85, 10.

Appropriate bridging molecules 2 for the use of the invention arecommercially available, or may easily be prepared according toprocedures known to those skilled in the relevant art. Suitable examplesmay for instance comprises an amine of formula —NH₂R₂ or diamine offormula —NH(R₂)—(CH₂)_(r)—NH(R′₂)— (in which r, R₂, R′₂ are as definedfor compounds of formula (I)), or suitable functional derivative thereofthat are commercially available or may be easily be obtained accordingto synthetic procedure known to those skilled in the relevant art.

Examples of specific procedures for the preparation of protectedbridging molecules 2, their coupling with the appropriate substratemolecule 1, and optional conversion of the obtained intermediates to thedesired compound of formula (I) are provided in the experimentalsection, together with relevant operational details.

As a general reference on possible protecting groups, and cleavageconditions, e.g. to implement the step e) of the above general syntheticprocedure, see the above cited “T. W. Green and P. G. M. Wuts;Protective groups in organic synthesis” Wiley 3^(rd) Ed. Chapters 5 and7.

The complexation of the compounds of formula (I) e.g. obtained from stepf) of former general preparation scheme with a paramagnetic ion and,particularly, with gadolinium, may be performed, for instance, bystoichiometric addition of a suitable Gd(III) derivative, particularly aGd(III) salt or oxide, to a solution of the ligand, e.g. by workingaccording to well-known experimental methods, for instance as reportedin EP 230893.

Finally, optional salification of the compounds of the invention may becarried out by properly converting any of the free acidic groups (e.g.carboxylic, phosphonic or phosphinic) or free amino groups into thecorresponding pharmaceutically acceptable salts. In this case too, theoperative conditions being employed for the optional salification of thecompounds of the invention are all within the ordinary knowledge of theskilled person.

Exemplificative implementation of the above general procedure leading tothe compounds of the formula (I) and of the chelate complexes thereof,are schematized herein below.

For instance, dimeric compounds according to the invention mayconveniently be prepared by using the synthetic procedure schematized inthe following general Scheme 1

in which the bis-epoxide 2 is reacted with two units of substrate 1A togive an intermediate 3 in which the nitrogen atom (of the bridgingmoiety) is in a protected form which is first deprotected and thenalkylated with the appropriate R₂ group to give the protected dimer offormula (II) that after cleavage of carboxy-protecting groups iscomplexed with the gadolinium metal ion to give the desired bis-Gdcomplex of formula (I).

Compounds of formula (IV) comprising a bridging molecule interrupted bytwo nitrogen atom may be analogously obtained, by using a correspondingbis-epoxide 2 comprising two suitably protected or alkylated nitrogenatoms.

Dimeric compounds of formula (I) may alternatively be prepared by usingthe synthetic procedure schematized in the following Scheme 2

According to this approach, a suitably protected Substrate 1B

is first obtained, e.g. by reaction of the commercially availableepichlorydrin with the substrate 1A, as described in details in theexperimental section, which is then reacted with the appropriated amineR₂NH₂ leading to the protected compound of formula 3 that is thendeprotected and complexed as above said.

Compounds of formula (IV) comprising a bridging molecule interrupted bytwo substituted nitrogen atoms may be analogously obtained by using theappropriate bis-amine e.g. of formula NH(R₂)(CH₂)rNH(R₂).

Specific examples of preparation of preferred compounds of formula (I)according to the invention are moreover provided in the followingexperimental section, constituting a general reference to the operativeconditions being employed in the above processes.

Dimers of formula (I) according to the present invention include twotetraaza macrocycles each having a hydroxylated residue on a nitrogenatom of the macrocyclic cage linked to one another by means of abridging moiety comprising one or more amine —NR₂— group(s).

Dimeric paramagnetic complexes according to the invention, having thesepeculiar structural components have interestingly proved to display ahigh relaxivity.

Relaxivity r_(1p) values measured for some representative complexcompounds of formula (I) are provided in Table A of the experimentalsection, by comparison with r_(1p) values measured, at the sameconditions, for some known MRI contrast agents currently used in thediagnostic daily practice, e.g. including Gd-DOTA, marketed as DOTAREM®,and Gd-HPDO3A marketed as ProHance®. By definition, relaxivity data,hence including those of the table A, are expressed in terms ofgadolinium concentration (mM).

Interestingly, relaxivity r_(1p) values measured for the dimeric complexcompounds of the invention are at least to 2 times higher than thatrecorded for commercial contrast agent of the marker (at the samegadolinium concentration).

In particular, the paramagnetic complex compounds of the formula (I) ofthe invention display a relaxivity r_(1p) value measured in humanplasma, at 37° C. and approx. 1.4 T which is of at least about 6,preferably higher than 7, and more preferably, higher than 8 mM⁻¹ s⁻¹.

Moreover, the paramagnetic complex compounds of the invention haveproven to display a low if not negligible protein binding with humanplasma proteins, including, for instance, the HSA.

In addition, the Applicant has observed that the presence of ahydroxylated pendant arm on each macrocyclic cage constituting thedimeric compounds of the invention, beside leading to complex compoundshaving favorable relaxivity and solubility, may also contribute toobtain aqueous solutions of corresponding complex paramagnetic endowedwith optimized viscosity. Advantageously, the high relaxivity displayedby the agents of the invention may allow to reduce their diagnosticallyeffective dose, as compared to current contrast agents. Paramagneticcomplexes and, especially, gadolinium complexes of the compounds offormula (I), or the pharmaceutical acceptable salt thereof, thus findadvantageous use in the preparation of pharmaceutical formulationsintended for a general use in the diagnostic imaging of a human oranimal body organ, tissue or region either in vivo or in vitro, ex vivo.

According to a further aspect, the invention relates to the use of thecompounds of formula (I) in the form complexes with a paramagnetic metalion and, especially, gadolinium, or of a pharmaceutical acceptable saltthereof, for the preparation of a pharmaceutical formulation for use inthe diagnostic imaging, either in vivo or in vitro, ex vivo, of a humanor animal body organ, tissue or region or of a biological sample,including cells, biological fluids and biological tissues originatingfrom a live mammal patient, and preferably, human patient, by use of theMRI technique.

A further aspect of the invention concerns a pharmaceutical compositionfor diagnostic use comprising a compound of formula (I) in the form ofparamagnetic metal complex or of a pharmaceutical salt thereof, inadmixture with one or more physiologically acceptable excipients,diluents or solvents. Preferably, the pharmaceutical composition is acontrast-producing composition and, more preferably, a MRI contrastproducing composition comprising at least one Gd-complex according tothe invention.

In an additional aspect the invention relates to a MRI contrast mediumcomprising an effective amount of at least one chelated compoundaccording to the invention and, especially, of a gadolinium complex ofthe formula (I), or of a pharmaceutical acceptable salt thereof, incombination with one or more pharmaceutically acceptable excipients,diluents or solvents.

To this extent, and unless otherwise provided, the term “effectiveamount” or “effective dose”, as used herein, refers to any amount of aparamagnetic chelated complex of the formula (I) according to theinvention or pharmaceutical composition thereof, that is sufficient tofulfil its intended diagnostic purpose(s): i.e., for example, to ex vivovisualize a biological element including cells, biological fluids andbiological tissues or the in vivo diagnostic imaging of body organs,tissues or regions of a patient.

Unless otherwise indicated, with “individual patient” or “patient” asused herein we refer to a living human or animal patient, and,preferably a human being undergoing MR diagnostic assessment.

Details concerning dosages, dosage forms, modes of administration,pharmaceutically acceptable carriers, excipients, diluents, adjuvantsand the like are known in the art.

Interestingly, and as above discussed, suitable dosage of theparamagnetic complexes according to the invention, i.e. allowing toobtain a diagnostically effective visualization of the body organ,tissue or region at least comparable to that obtained in the dailypractice with the MRI contrast agents of the market, may include anamount of the paramagnetic complex lower than that currently used withNon-Specific contrast agents of the market.

For instance, satisfactory diagnostic MRI images, providing a physicianwith adequate diagnostic support, may be obtained with doses of thegadolinium complex compounds identified by the present invention ofabout 90%, more preferably 80%, and up to 60% of the dose of MRIcontrast agent used in the daily practice, which for adult patientscommonly is of about 0.1 mmol/kg of patient body weight.

From all the foregoing it can be easily envisaged that the selection ofparamagnetic complex compounds of formula (I) identified by the presentinvention have a wide range of applications as they can be used forintravasal, (for instance intravenous, intraarterial, intracoronaric,intraventricular administration and the like), intrathecal,intraperitoneal, intralymphatic and intracavital administrations.Furthermore, they are suitable for the oral or parenteral administrationand, therefore, specifically for the imaging of the gastrointestinaltract.

For instance, for parenteral administration they can be preferablyformulated as sterile aqueous solutions or suspensions, whose pH canrange from 6.0 to 8.5.

These formulations can be lyophilized and supplied as they are, to bereconstituted before use.

For the gastrointestinal use or for injection in the body cavities,these agents can be formulated as a solution or suspension optionallycontaining suitable excipients in order, for example, to controlviscosity.

For the oral administration they can be formulated according topreparation methods routinely used in the pharmaceutical technique or ascoated formulations to gain additional protection against the stomachacidic pH thus preventing, in case of chelated metal ions, their releasewhich may take place particularly at the typical pH values of gastricfluids.

Other excipients, for example including sweeteners and/or flavouringagents, can also be added, according to known techniques ofpharmaceutical formulations.

The solutions or suspensions of the compounds of this invention can alsobe formulated as aerosol to be used in aerosol-bronchography andinstillation.

For example, they can be also encapsulated into liposomes or evenconstitute the liposomes themselves, as set forth above, and thus can beused as uni- or multi-lamellar vesicles.

In a preferred aspect, pharmaceutical compositions according to theinvention are properly formulated in isotonic sterile aqueous,optionally buffered, solutions for parenteral administration, and mostpreferably for intravenous or intra-arterial administration.

More preferably, the said diagnostic composition has a concentration ofthe paramagnetic complex of the formula (I) of from 0.002 and 1.0 M andis supplied, for instance as a bolus, or as two or more doses separatedin time, or as a constant or non-linear flow infusion.

In a further aspect, the invention relates to the use of apharmaceutical composition including a paramagnetic chelated complex ofthe formula (I) or pharmaceutical acceptable salt thereof for thediagnostic imaging, both in vitro (ex vivo) and in vivo, of pathologicalsystems, including cells, biological fluids and biological tissuesoriginating from a live mammal patient, and preferably, human patient,as well as of human body organ, regions or tissues, including tumors orcancerous tissues, inflammations, as well as for monitoring the progressand results of therapeutic treatment of the said pathologies.

In an additional aspect, the present invention concerns a method for thein vivo imaging of a body organ, tissue or region by use of the MRItechnique, said method comprises enhancing the signal generated by thewater protons by use of a paramagnetic chelated complex of the formula(I) according to the invention, or a physiological acceptable saltthereof.

In one embodiment, said method comprises administering to a human oranimal patient to be imaged a diagnostically effective amount of acomposition of the invention comprising a compound of formula (I) in theform of complex with a paramagnetic metal ion, and, preferably, with theGd³⁺ metal ion and then subjecting the administered patient to thediagnostic imaging by use of the MRI technique.

According to a particularly preferred embodiment, the above MRI methodis instead performed on human or animal bodies suitably pre-administeredwith a diagnostically effective amount of a composition of the inventionas above defined.

More particularly, according to a preferred embodiment the presentinvention refers to a method for the in vivo imaging a human or animalbody organ or tissue by use of the MRI technique that comprises thesteps of:

a) submitting a human or animal pre-administered with a composition ofthe invention comprising a compound of formula (I) in the form of aparamagnetic complex, or of a pharmaceutically acceptable salt thereof,and positioned in a MRI imaging system, to a radiation frequencyselected to excite the non-zero proton spin nuclei of the activeparamagnetic substrate; and

b) recording a MR signal from said excited nuclei.

In yet another aspect the invention provides a method for the in vitro(ex vivo) imaging of biological samples, including cells, biologicalfluids and biological tissues originating from a live mammal patient,and preferably, human patient, by use of the MRI technique, thatcomprises contacting an effective amount of a paramagnetic complexcompound of formula (I), or of a physiologically acceptable saltthereof, with the biological sample of interest and then obtaining MRIsignals from said samples by use of the MRI technique.

Non-limiting examples of preferred compounds of the invention andintermediates for their preparation is reported in the followingsection, aimed to illustrate the invention in greater detail withoutlimiting its scope.

EXPERIMENTAL PART Example 1: Preparation of the Substrate 1B

This compound was obtained by using the synthetic procedure shown inScheme 3:

comprising:

a) Preparation of Compound 1B

Commercially available epichlorohydrin 2 (10.5 mL; 137 mmol) wasdissolved in acetonitrile (300 mL) and the resulting solution was slowlyadded at room temperature to a solution of DO3A tris-t-butyl ester 1A(Org. Synth. 2008, 85, 10) (14.1 g; 27.4 mmol) in acetonitrile (100 mL).The mixture was stirred for 24 h then more epichloridrin 2 (5.2 mL; 68mmol) was added. After 24 h the mixture was evaporated and the residuepurified by chromatography on silica gel (eluent: CH₂Cl₂/MeOH=50:1→1:1)to give compound 1C (10.6 g). Yield 64%.

1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

Example 2: Preparation of the Chelate Complex 1

This dimeric compound was prepared using the procedure of the followinggeneral Scheme 4:

including:

a) Preparation of 1

Benzyl chloroformate (95%; 18.85 g; 105 mmol) was added in 1 h to amixture of diallylamine (commercially available) (9.7 g; 100 mmol),K₂CO₃ (34.5 g; 250 mmol), water (150 mL) and EtOAc (150 mL) at 0° C.After stirring for 6 h, the organic phase was separated and extractedwith 1 N HCl (2×100 mL), water (100 mL) and brine (100 mL). The organicphase was dried (Na₂SO₄) and evaporated to give 1 (22 g). Yield 95%.1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

b) Preparation of Protected Bridging Molecule 2

A solution of 3-chloroperbenzoic acid (MCPBA) (75%; 34.5 g; 150 mmol) indichloromethane (100 mL) was added dropwise to a solution ofintermediate 1 (11.6 g; 50 mmol) in dichloromethane (100 mL). Thesolution was stirred at room temperature for 16 h. More MCPBA (11.5 g)was added and the mixture stirred for other 48 h. The mixture wasfiltered, washed with 10% aq. Na₂SO₃ (2×100 mL), 5% aq. NaHCO₃ (4×100mL), H₂O (100 mL) and brine (100 mL). The organic phase was separated,evaporated and the residue purified by chromatography on silica gel(eluent: n-heptane/EtOAc=2:1) to obtain 2 (11.7 g). Yield 89%.

1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

c) Preparation of Intermediate 3

A solution of Substrate 1A (Org. Synth. 2008, 85, 10) (43.2 g; 84 mmol),intermediate 2 (10 g; 38 mmol) and N,N-diisopropylethylamine (DIPEA)(216 g; 1.68 mol) in acetonitrile (500 mL) was stirred at 60° C. for 48h. The mixture was evaporated to a residue which was dissolved in EtOAc(300 mL). The solution was washed with water (4×100 mL), brine (4×100mL), filtered and evaporated to a residue that was purified by flashchromatography on silica gel (eluent: EtOAc/MeOH=1:1) to giveintermediate 3 (30 g). Yield 61%.

1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

d) Preparation of Intermediate 4

Palladium 5% carbon (wet with about 50% water) (5 g) was added to asolution of intermediate 4 (25 g; 19.3 mmol) in MeOH (300 mL). Themixture was stirred and hydrogenated at room temperature and atmosphericpressure for 8 h. The mixture was filtered and evaporated to giveintermediate 4 (21.5 g). Yield 96%.

1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

e) Preparation of Protected Ligand 5

A solution of t-butyl bromoacetate (3.7 g; 19 mmol) in acetonitrile (50mL) was added in 30 min to a mixture of compound 5 (20 g; 17.3 mmol) andK₂CO₃ (5.53 g; 40 mmol) in acetonitrile (200 mL). The mixture wasstirred for 48 h at room temperature then filtered and evaporated. Theresidue was purified by chromatography on silica gel (eluent: gradientof EtOAc/MeOH) to give 5 (19.4 g). Yield 88%.

1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

f) Preparation of Ligand 6

Trifluoroacetic acid (19 mL) was added to a solution of intermediate 6(15.3 g; 12 mmol) in dichloromethane (70 mL) at 0° C. The mixture wasstirred for 6 h then evaporated; the residue was dissolved in TFA (80mL) and triisopropylsilane (0.5 mL) was added. The mixture was stirredat room temperature for 16 h, then evaporated. The solid was purified bychromatography on Amberchrome CG161M column (eluent: gradient ofwater/MeCN) obtaining the chelating ligand 6 as a solid (8.76 g). Yield83%.

1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

g) Complexation

Gadolinium chloride hexahydrate (3.38 g, 9.1 mmol) was added to asolution of chelating ligand 7 (8 g; 9.1 mmol) in water (100 mL) and thepH of the mixture was slowly increased to pH 6.5-7 with 1 N NaOH. Theobtained solution was stirred at room temperature for 5 h then filteredon Millipore HA 0.45 μm, concentrated and purified by chromatography onAmberchrome CG161M column (eluent: gradient of water/MeCN) obtaining10.1 g of the corresponding gadolinium complex. Yield 92%.

Mass spectrum and elemental analysis were consistent with the expectedstructure.

Applying the same synthetic strategy and employing the triflate ofhydroxymethylphosphonate di-t-butyl ester (synthesized as reported inUS2014/0086846, page 33) the Chelate Complex 2 was prepared.

Example 3: Preparation of the Chelate Complex 3

This complex compound was obtained by using the procedure shown inScheme 5:

Including:

a) Preparation of 2

Commercially available epichlorohydrin (4.1 mL; 52 mmol) was added to asolution of commercially available D-glucamine 1 (1.9 g; 10.5 mmol) inMeOH (110 mL). The mixture was stirred at 50° C. for 26 h thenevaporated to give the bridging molecule 2 as a colourless oil that wasdirectly used for the next reaction without any further purification.Quantitative yield.

1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

b) Preparation of 3

A solution of Substrate 1A (Org. Synth. 2008, 85, 10) (10.7 g; 21 mmol)in acetonitrile (14 mL) was added to a solution of compound 2 (3.8 g;10.5 mmol) in DMSO (14 mL) and Et₃N (4.3 mL). The mixture was stirred at70° C. for 72 h then evaporated. The residue was purified bychromatography on Amberlite XAD 1600 (eluent: gradient of water/MeCN) togive the protected ligand 3 (2.1 g). Yield 15%.

1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

c) Preparation of Ligand 4

Trifluoroacetic acid (1.1 mL) was added to a solution of 3 (2.1 g; 1.6mmol) in dichloromethane (30 mL). The mixture stirred for 30 min thenwas evaporated. The residue was dissolved in TFA (3.7 mL) andtriisopropylsilane (0.1 mL) was added. The obtained mixture was stirredfor 24 h at room temperature then evaporated and the residue purified bychromatography on Amberlite XE 750 column (eluent: gradient ofwater/MeCN) obtaining the desired ligand 4 (1.5 g). Yield 95%.

1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

d) Complexation

Ligand 4 (1.5 g; 1.5 mmol) was dissolved in water (20 mL), gadoliniumchloride hexahydrate (1.13 g; 3 mmol) was added then 1M NaOH was addedto achieve pH 7. The mixture was stirred at 50° C. for 6 h. The solutionwas then filtered on Millipore HA 0.25 μm filters and evaporated underreduced pressure. The crude product was purified on Amberchrome CG161Mcolumn (eluent: gradient of water/acetonitrile). The fractionscontaining the pure product were pooled and evaporated. The solidproduct was dried under vacuum to obtain the gadolinium complex as awhite powder (1.4 g). Yield 72%.

Mass spectrum and elemental analysis were consistent with the expectedstructure.

Applying the same synthetic strategy and employing the2-[2-(aminomethyl)-3-[2-hydroxy-1-(hydroxymethyl)ethoxy]propoxy]-1,3-propanediol(prepared for instance as reported in Chem. Commun. 2005, 474-476) theChelate Complex 8 was prepared.

Example 4: Preparation of the Chelate Complex 5

This complex compound was obtained by using the procedure shown inScheme 6:

including:

a) Preparation of 2

Epichlorohydrin (3.7 g; 40 mmol) was added to a solution of 1 (preparedas reported in Tetrahedron 2010, 66, 8594-8604) (2 g; 7 mmol) in MeOH(40 mL). The reaction mixture was stirred at room temperature for 56 h.The white solid precipitated was filtered and dried to give compound 2(3.28 g). Yield 55%. 1H-NMR, 13C-NMR and mass spectrum were consistentwith the expected structure.

b) Preparation of Protected Ligand 3

Substrate 1A (Org. Synth. 2008, 85, 10) (15 g; 29 mmol) was added to asolution of compound 2 (4.2; 8.9 mmol) and Et₃N (3.6 g; 36 mmol) in MeCN(60 mL). The mixture was stirred at 50° C. for 48 h then at 70° C. for20 h. The mixture was evaporated, the residue treated with EtOAc (100mL) and filtered. The organic phase was washed with water (2×100 mL),brine (2×100 mL) then evaporated. The residue was purified by flashchromatography on silica gel (eluent: CH₂Cl₂/MeOH=100:1→1:1) to give theprotected ligand 3 as pale yellow oil (4.55 g). Yield 36%.

1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

c) Preparation of Ligand 4

Trifluoroacetic acid (6 mL; 48 mmol) and triisopropylsilane (0.1 mL)were added to compound 3 (4.5 g, 3 mmol). The solution was stirred atroom temperature for 24 h. The solvent was evaporated and the residuepurified by chromatography on Amberlite XE 750 column (eluent: gradientof water/MeCN) obtaining the desired ligand 4 (3 g). Yield 96%.

1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

d) Complexation

Ligand 4 (3 g; 3 mmol) was suspended in water (60 mL) and gadoliniumchloride hexahydrate (2.27 g; 6.1 mmol) was added. 1M NaOH was added toachieve pH 7 and the homogeneous solution was stirred at 50° C. for 2 h.The solution was then filtered on Millipore HA 0.25 μm filters andevaporated under reduced pressure. The crude product was purified onresin Amberchrome CG161M column (eluent: water/acetonitrile). Thefractions containing the pure product were pooled and evaporated. Thesolid product was dried under vacuum to obtain the gadolinium complex asa white powder (2 g). Yield 49%.

Mass spectrum and elemental analysis were consistent with the expectedstructure.

Example 5: Preparation of the Chelate Complex 7

This complex compound was obtained by using the procedure shown inScheme 7:

a) Preparation of 2

Epichlorohydrin (2.8 mL; 36 mmol) was added to a solution ofcommercially available benzylamine 1 (1.64 g; 15 mmol) in EtOH (10 mL).The mixture was stirred at room temperature for 30 h then evaporated togive the protected bridging molecule 2 that was directly used for thenext reaction without any further purification. Quantitative yield.

1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

b) Preparation of Intermediate 3

A solution of substrate 1A (Org. Synth. 2008, 85, 10) (15.4 g; 30 mmol)in MeCN (30 mL) was added to a solution of compound 2 (438 g; 15 mmol)in MeCN (30 mL) and Et₃N (6.3 mL). The mixture was stirred at 55° C. for96 h then evaporated. The residue was purified by flash chromatographyon silica gel (eluent: CH₂Cl₂/MeOH=100:1→1:1) to give intermediate 3 (10g). Yield 53%.

1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

b) Preparation of 4

A solution of intermediate 3 (10 g; 8 mmol) in methanol (80 mL) wasadded with 5% palladium on carbon (wet with about 50% water) (2.5 g) andhydrogenated at 45° C. for 5 h. More catalyst (0.8 g) was added and themixture hydrogenated at 45° C. for other 4 h The catalyst was filteredand the solution evaporated to give intermediate 4 (8.9 g). Yield 96%.

1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

c) Preparation of the Protected Ligand 6

Tetraethylene glycol monotosylate 5 (2.6 g, 7.5 mmol) (commercialproduct, e.g. Aldrich) was added to a solution of 4 (8.5 g; 7.3 mmol) inMeCN (mL) and the mixture was stirred for 72 h. The mixture wasevaporated, the residue dissolved in CHCl₃ (200 mL) and washed withwater (2×100 mL). The organic phase was separated, dried and evaporated.The residue was purified by flash chromatography on silica gel (eluent:CH₂Cl₂/MeOH=100:1→1:1) to give the protected ligand 6 (8.2 g). Yield88%.

1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

d) Preparation of the Ligand 7

Trifluoroacetic acid (5 mL) was added to a solution of intermediate 6 (8g; 6.3 mmol) in dichloromethane (50 mL). The mixture stirred for 30 minthen was evaporated. The residue was dissolved in TFA (20 mL) andtriisopropylsilane (0.1 mL) was added. The obtained mixture was stirredfor 24 h at room temperature then evaporated and the residue purified bychromatography on Amberlite XE 750 column (eluent: gradient ofwater/MeCN) obtaining the desired ligand 7 (5.3 g). Yield 84%.

1H-NMR, 13C-NMR and mass spectrum were consistent with the expectedstructure.

d) Complexation

Ligand 7 (4.5 g; 4.5 mmol) was dissolved in water (100 mL), gadoliniumchloride hexahydrate (1.7 g; 4.6 mmol) was added then 1M NaOH was addedto achieve pH 7. The mixture was stirred at 50° C. for 18 h. Thesolution was then filtered on Millipore HA 0.25 μm filters andevaporated under reduced pressure. The crude product was purified onAmberchrome CG161M column (eluent: gradient of water/acetonitrile). Thefractions containing the pure product were pooled and evaporated. Thesolid product was dried under vacuum to obtain the gadolinium complex asa white powder (4.4 g). Yield 75%.

Mass spectrum and elemental analysis were consistent with the expectedstructure.

Applying the same synthetic strategy and employing the2-(2-hydroxyethoxy)ethyl 4-methylbenzenesulfonate (commerciallyavailable) the Chelate Complex 4 was prepared.

Example 6: Relaxometric Properties

The relaxometric properties of some representative complex compoundsaccording to the invention have been determined at different magneticfield strengths, e.g. including 0.47 and 1.41 T, at 37° C. and indifferent media (physiologic solution and human plasma) and comparedwith relaxivity values measured, at the same conditions, for someGd-Complex of the market having an analogous cyclic coordination cage.

Materials

Apparatus

The longitudinal water proton relaxation rate (R₁=1/T1) was measured at0.47 T with a Minispec MQ-20 spectrometer (Bruker Biospin, Germany)operating at a proton Larmor frequency of 20 MHz; MR experiments at 1.41T were performed using a Minispec MQ-60 spectrometer (Bruker Biospin,Germany) operating at a proton Larmor frequency of 60 MHz.

Methods

Sample Preparation

All test articles were used as supplied and diluted in the selectedmedium (physiologic solution or human plasma) by weighting the requiredamount of paramagnetic chelated complex to get a 5 or 10 mM startingsolution.

Relaxivity Measurements

Five different concentration samples (0.1, 0.25, 0.5, 0.75 and 1 mM) foreach medium have been prepared by further dilution of the starting 5 or10 mM solution.

Relaxation Measurement

Relaxivity measurements were performed at 0.47 T and 1.41 T at a presettemperature sample of 37° C., kept constant by means of a thermostaticbath connected to the sample holder of the spectrometer. The five samplesolutions have been preliminary pre-heated at 37° C. in an externalthermostatic bath and then left 10 minutes inside the internal bath toassure the stabilization of the temperature. Longitudinal relaxationtime T₁ was measured by means of a standard inversion recovery sequence,where the inversion time (TI) was varied from 10 ms to at least 5 timesT₁ in 15 steps. Statistical analysis (mono-exponential fitting for T₁measurement, linear fitting for the evaluation of longitudinalrelaxivity) was performed by Mathematica® (Wolfram, USA). Errors on theestimated parameters were evaluated by the fitting procedure.

Results

The relaxivity values r_(1p) obtained from some representative compoundsaccording to the invention, both in physiologic solution and in humanplasma, at 37° C., are summarized in the following Table A, togetherwith the structure of tested compounds and the strength of the appliedmagnetic field (in T), and compared with corresponding values measuredfor some commercial contrast agents in clinical practice.

By definition, relaxivity data, and hence including those of the tablebelow, are expressed in terms of gadolinium concentration.

TABLE A r_(1p) [mM⁻¹s⁻¹] r_(1p) at 0.47 r_(1p) at 1.41 T 37° C., T 37°C., r_(1p) at 0.47 T human r_(1p) at 1.41 T human Complex 37° C., salineplasma 37° C., saline plasma

3.6 4.5 3.2 3.6

3.5 4.9 3.1 4.15

8.3 9.7 8.5 9.2

9.0 12.0 9.3 11.3

9.3 11.5 9.4 10.8

7.3 10.6 7.5 10.2

CONCLUSIONS

The relaxivity of the investigated contrast agents ranges between 3.5(for Prohance®) and 9.0 (for the Chelate Complex 2) mM⁻¹ s⁻¹ at 0.47 Tin physiologic solution, and from 4.9 to 12.0 mM^(−l)s⁻¹ in plasma, samemagnetic field, same mM Gd³⁺ concentration. These results confirm thatthe particular selection represented by the paramagnetic complexes and,especially, the Gd³⁺ complexes of the compounds of formula (I) of theinvention show an increased relaxivity r_(1p), which is at least about 2times the relaxivity shown, at the same conditions (i.e. in saline or inhuman plasma, at 37° C.), by the Non Specific contrast agents currentlyin use in the daily diagnostic practice, such as Dotarem® and ProHance®.

1. A compound of formula (I)

where: R is —CH(R₁)—COOH, where: R₁ is H or a C₁-C₃ alkyl chain that isoptionally substituted by a C₁-C₃ alkoxy or C₁-C₃ hydroxyalkoxy group; nis 1 or 2; R₂ is selected from the group consisting of: an aryl ring; acyclohexyl ring; a C₁-C₅ alkyl substituted by one or more C₁-C₈hydroxyalkoxy groups, or by a cycloalkyl ring; a group of formula—(CH₂)_(s)CH(R₃)-G; and a C₅-C₁₂ hydroxyalkyl comprising at least 2hydroxyl groups; in which s is 0, 1 or 2; R₃ is H, or an arylalkylene orcycloalkyl-alkylene having from 1 up to 3 carbon atoms in the alkylenechain; G is a group selected from —PO(OR₄)₂, —PO(R₅)(OR₄) and —COOH; inwhich R₄ independently of one another is H or C₁-C₅ alkyl; R₅ is an arylor a cyclohexyl ring, or C₁-C₅ alkyl which is optionally substituted byan aryl or cyclohexyl ring; and L is a C₁-C₆ alkylene, optionallyinterrupted by one or more —N(R′₂)— groups, and optionally substitutedby one or more substituent groups selected from hydroxyl, C₁-C₃ alkoxyand C₁-C₃ hydroxyalkoxy, where R′₂ is, independently, as defined for R₂,as well as individual diastereoisomers and their racemic mixtures,geometric isomers and solved enantiomers of the same, and thephysiologically acceptable salt thereof.
 2. The compound according toclaim 1 in which R₁ is H.
 3. The compound according to claim 1 in whichL is C₁-C₆ alkylene, having the following formula (II)

in which: m is 1, 2, 3, 4, 5, or 6; and n and R₂ are as defined inclaim
 1. 4. (canceled)
 5. (canceled)
 6. (canceled)
 7. (canceled)
 8. Thecompound according to claim 3 in which, in the formula (II), R₂ is agroup of formula —(CH₂)_(s)CH(R₃)-G.
 9. The compound according to claim8 in which R₃ is H, having the formula (II B)

in which: s is 0, 1 or 2; n and m, independently to one another, are 1or 2; and G is a group selected from —PO(OR₄)₂, —PO(R₅)(OR₄) and —COOH,in which R₄ is H or a tert-butyl; and R₅ is an optionally substitutedphenyl or cyclohexyl ring, or a C₁-C₃ alkyl substituted or not by aphenyl or cyclohexyl ring.
 10. The compound according to claim 9 inwhich in the formula (II B) s is 0 or 1; n and m are both 1; and, G isselected from —PO(OH)₂ and —COOH.
 11. The compound according to claim 3in which, in the formula (II), R₂ is a C₁-C₅ alkyl substituted by one ortwo C₁-C₅ hydroxyalkoxy group(s) or by a cycloalkyl ring.
 12. Thecompound according to claim 11 in which R₂ is a C₂-C₁₀hydroxyalkoxy-alkylene selected from the group consisting of formula—CH₂(OCH₂CH₂)_(s)OCH₂OH, —CH₂(CH₂OCH₂)_(r)CH₂OH and—(CH₂)_(r)—O(CH₂)_(r)OH, where r is 1 or 2 and s is as defined inclaim
 1. 13. The compound according to claim 11 in which R₂ is a C₁-C₅alkyl substituted by a saturated C₅-C₇ cycloalkyl ring.
 14. The compoundaccording to claim 13 in which R₂ is a C₁-C₅ alkyl substituted by acyclohexyl ring.
 15. The compound according to claim 13 in which R₂ is aC₁-C₅ alkyl substituted by a piperidine, or a piperidine derivativehaving from 1 to 8 substituents groups linked to the carbon atom(s) ofthe heterocyclic ring.
 16. The compound according to claim 1 in which inthe formula (I) L is a C₁-C₆ alkylene chain interrupted by one or two—N(R′₂)— groups, having the formula (III)

in which: each n, r and d is, independently, 1 or 2; and R₂ and R′₂,equal or different, are as defined in claim
 1. 17. The compoundaccording to claim 16 in which: d is 1; and R′₂=R₂, having the formula


18. The compound according to claim 17 in which, in the formula (IV), R₂is a group of formula —(CH₂)_(s)CH(R₃)-G where s, R₃ and G are asdefined in claim
 1. 19. The compound according to claim 18, of formula(IV A)

in which n is 1; r is 1 or 2; s is 0, 1 or 2; and G is a group selectedfrom —PO(OH)₂ and —COOH.
 20. The compound according to claim 1 offormula:


21. A chelated complex of a compound according to claim 1 with twoparamagnetic metal ions selected from the group consisting of Fe²⁺,Fe³⁺, Cu²⁺, Cr^(3+,) Gd³⁺, Eu³⁺, Dy³⁺, La³⁺, Yb³⁺ and Mn²⁺, or aphysiologically acceptable salt thereof.
 22. The chelated complexaccording to claim 21, wherein the paramagnetic metal ions are Gd³⁺ions.
 23. The compound according to claim 1, wherein the physiologicallyacceptable salt is with a cation of (i) an inorganic base selected froman alkali metal and alkaline-earth metal, (ii) an organic base selectedfrom ethanolamine, diethanolamine, morpholine, glucamine,N-methylglucamine, and N,N-dimethylglucamine or (iii) an amino acidselected from lysine, arginine and ornithine.
 24. The chelated complexaccording to claim 21, wherein the physiologically acceptable salt iswith a cation of (i) an inorganic base selected from an alkali metal andalkaline-earth metal, (ii) an organic base selected from ethanolamine,diethanolamine, morpholine, glucamine, N-methylglucamine, andN,N-dimethylglucamine or (iii) an amino acid selected from lysine,arginine and ornithine.
 25. A method of MR imaging comprising:administering the chelated complex as defined in claim 21 to a patient;submitting the patient to a radiation frequency selected to excitenon-zero proton spin nuclei of the chelated complex; and recording a MRsignal from said nuclei.
 26. A pharmaceutical composition comprising achelated complex of claim 21 and at least one of a pharmaceuticallyacceptable carrier, a diluent an excipient and combinations thereof. 27.A compound as defined in claim 1 in which each of the carboxylic groupsR linked to the nitrogen atoms of the macrocycle is in a protected formas tert-butyl ester.